Pancreatic Cancer-Related Mutational Burden Is Not Increased in a Patient Cohort With Clinically Severe Chronic Pancreatitis

Abstract

Introduction: Chronic pancreatitis is associated with an increased risk of developing pancreatic cancer, and patients with inherited forms of pancreatitis are at greatest risk. We investigated whether clinical severity of pancreatitis could also be an indicator of cancer risk independent of etiology by performing targeted DNA sequencing to assess the mutational burden in 55 cancer-associated genes.

Methods: Using picodroplet digital polymerase chain reaction and next-generation sequencing, we reported the genomic profiles of pancreases from severe clinical cases of chronic pancreatitis that necessitated palliative total pancreatectomy with islet autotransplantation.

Results: We assessed 57 tissue samples from 39 patients with genetic and idiopathic etiologies and found that despite the clinical severity of disease, there was no corresponding increase in mutational burden. The average allele frequency of somatic variants was 1.19% (range 1.00%-5.97%), and distinct regions from the same patient displayed genomic heterogeneity, suggesting that these variants are subclonal. Few oncogenic KRAS mutations were discovered (7% of all samples), although we detected evidence of frequent cancer-related variants in other genes such as TP53, CDKN2A, and SMAD4. Of note, tissue samples with oncogenic KRAS mutations and samples from patients with PRSS1 mutations harbored an increased total number of somatic variants, suggesting that these patients may have increased genomic instability and could be at an increased risk of developing pancreatic cancer.

Discussion: Overall, we showed that even in those patients with chronic pancreatitis severe enough to warrant total pancreatectomy with islet autotransplantation, pancreatic cancer-related mutational burden is not appreciably increased.

Figure 1.

Characteristics of TPIAT patient cohort. (a) Proportion of all patient cases according to sex, age at onset (pediatric younger than 19 years), and etiology. (b) Proportion of genetic cases only, classified by contributing gene mutations and age at onset. (c) Age at onset of idiopathic cases only. (d) Total patients according to the highest PanIN grade identified in tissue sections. (e) Representative hematoxylin and eosin–stained tissue section from a patient undergoing TPIAT (scale bar = 200 µm). PanIN, pancreatic intraepithelial neoplasia; TPIAT, total pancreatectomy with islet autotransplantation.

Figure 2.

Identification of somatic variants. (a) Total somatic variants per patient sample. (b) Total and (c) COSMIC somatic variants from all patient samples in relation to amplicon coverage for each gene. (d) Venn diagrams demonstrating overlap between FreeBayes and VarScan variant callers. COSMIC, Catalogue of Somatic Mutations in Cancer.

Figure 3.

COSMIC somatic variants in chronic pancreatitis tissue samples. Genes harboring 1 or more COSMIC somatic variants, as determined by our amplicon panel, are highlighted. Genes are sorted by prevalence of nonsynonymous variants. COSMIC, Catalogue of Somatic Mutations in Cancer; LOF, loss of function.

Figure 4.

KRAS mutations in TPIAT patient cohort. Graphs show mean ± SD. (a) Total somatic variants from KRAS mutation-bearing patients (p.G12R or p.Q61R) compared with other samples and (b) COSMIC somatic variants between those groups. (c) Fibrosis score and (d) disease duration revealed no discernable differences between KRAS mutation-bearing patients and other patients. (e) Representative Sanger sequencing results demonstrating enrichment of KRAS p.G12R variants in MN17E and MN25F DNA, but not MN24D DNA, on treatment with double-stranded nuclease (DSN) as part of nuclease-assisted minor allele enrichment with probe overlap analysis. (f) Representative digital droplet PCR confirmation of KRAS p.G12R variants in MN17E and MN25F samples and the absence of variants in MN35A using fluorescently labeled TaqMan probes that were optimized using PSN-1 and BxPC-3 cell lines that contain KRAS p.G12R or wild-type KRAS, respectively, and serve as a control. COSMIC, Catalogue of Somatic Mutations in Cancer; TPIAT, total pancreatectomy with islet autotransplantation.

Figure 5.

Single-nucleotide variant correlation with clinical characteristics. Total somatic variants and COSMIC somatic variants are compared (a) between female and male patients (mean ± SD); (b) with disease duration; (c) according to age at onset of disease (pediatric younger than 19 years; mean ± SD); (d) with age at TPIAT; (e) with patient fibrosis scores; (f) according to PanIN score (mean ± SD); and (g) according to the presence of calcification (mean ± SD). (h) For smokers, smoking duration is compared with total somatic variants. COSMIC, Catalogue of Somatic Mutations in Cancer; PanIN, pancreatic intraepithelial neoplasia; TPIAT, total pancreatectomy with islet autotransplantation.

Figure 6.

Increased mutational burden in patients with PRSS1 mutations. (a) Samples from PRSS1 mutation-bearing patients have a higher average number of somatic variants and COSMIC somatic variants than all other samples together (mean ± SD). (b) Samples from PRSS1 mutation-bearing patients also have increased total and COSMIC somatic variants compared with other hereditary pancreatitis samples harboring mutations in CFTR, CTRC, or SPINK1 and a higher average number of somatic variants than idiopathic cases. (c) Fibrosis scores and (d) disease duration show no differences among PRSS1 mutation-bearing patients, idiopathic cases, or other hereditary pancreatitis cases (mean ± SD). COSMIC, Catalogue of Somatic Mutations in Cancer.